Investigation of expression and localization of biomolecules (e.g., proteins) through the combination of immunohistochemistry (IHC) and light microscopy is a pivotal technique in diagnostic and basic research. Classic chromogenic IHC allows for 2-dimensional (2D) imaging of relatively thin tissue sections (e.g., 1-10 μm). Imaging of brain tissue in 3D is an essential technique to understand the cytoarchitecture and spatial relationship between cell populations. In general, processing and staining an organ and a tissue for 3D imaging provide a better understanding of complex biological processes. Specifically, understanding integrated 3D structure and fine molecular details throughout an organ or a tissue provide detailed insights into normal functions, and changes resulted from or giving rise to pathological states.
The introduction of fluorescent laser-scanning light microscopy in combination with imaging analysis software has allowed for 3-dimensional (3D) investigation of tissue architecture and protein localization in 10-50 μm sections. Before the introduction of clearing techniques, 3D imaging was limited to reconstructions of confocal stacks of thin sections, or digital reconstruction of macro-scale structures using annotation of multiple 2D images, a labor-extensive approach for partial or whole mouse brains1, and human tissue blocks2. The introduction of tissue clearing techniques3, beginning with the precursor CLARITY method4, has advanced 3D histology by enabling imaging of relatively thick tissue samples (100 μm to several mm). Clarification methods reduce sample opacity generated by lipid-driven light scattering and make the organ or tissue transparent. Lipids are removed with detergents and replaced with a hydrogel matrix, allowing better penetration of excitation light as well as undisturbed detection of emission light. Several clearing methods are currently available, differing by the applications, the chemicals, the device, the procedure length, the degree of transparency, and the cost3,5-7.
The current methods of processing and staining tissue exhibit several shortcomings. Some clearing methods, such as CLARITY®, require expensive equipment and fail to significantly improve staining beyond about 50 μm. While other methods provide for staining beyond 50 μm (e.g., 100 μm to greater than 2000 μm), application of those methods have been limited to animal models expressing a fluorescently-tagged protein, non-human (e.g., rat brain8-12), or non-complex tissues (e.g., tissues lacking the connective tissue present in adult tissues). Some methods also use reagents harsh to the samples, resulting in poor data quality.
Human tissues require immunohistochemistry (IHC) labeling. Particular challenges associated with fluorescent IHC of human tissues6,13-15 (e.g., brain) include variability in the procurement process, fixing techniques, preservation, and internal sources of auto-fluorescence. The recent emergence of various clarification methods5,14,16,17 allows post-mortem human brains to be cleared. IHC of thick samples, however, remains challenging. Due to the poor penetration of antibody molecules18,19, passive diffusion of antibodies leads to inconsistent results in such samples. Reproducibility also remains an issue14 of methods utilizing electric fields19 or system-wide binding controlling agents20. Thus, methods and devices that improve processing and/or staining of relatively thick tissues, especially penetration of labeling agents (e.g., antibody) are needed in 3D imaging.